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. 2014 Apr;21(4):444-9.
doi: 10.1038/gt.2014.5. Epub 2014 Feb 27.

Efficient Delivery of Lentiviral Vectors Into Resting Human CD4 T Cells

Free PMC article

Efficient Delivery of Lentiviral Vectors Into Resting Human CD4 T Cells

X Geng et al. Gene Ther. .
Free PMC article

Erratum in


Resting human CD4 T cells are highly resistant to transfection or infection with lentiviral vectors derived from the human immunodeficiency virus. We now describe a flexible and efficient approach involving virus-like particles containing simian immunodeficiency virus lentiviral gene product protein X and pseudotyping with CXCR4-tropic HIV Env. This method permits effective genetic manipulation of these cells while preserving their naturally quiescent state. This technology can also be extended to primary lymphoid cultures where authentic cellular composition and functional relationships are preserved.

Conflict of interest statement


The authors declare no conflict of interest.


Figure 1
Figure 1
Genetic manipulation of resting CD4 T cells by lentiviral vectors using sequential spinoculation of Vpx-VLP and shRNA-encoding HIV LV particles. (a) The spinoculation procedure. Fresh target cells were spinoculated with Vpx-VLPs, followed by shRNA-encoding HIV LV particles. Particles were pseudotyped with the CXCR4-tropic Env of HIV-1, which supports efficient fusion of virions to resting CD4 T cells. A detailed description is available in the Methods section (b) Spinoculation of Vpx-VLPs degrades SAMHD1 in resting CD4 T cells isolated from peripheral blood, tonsil, or spleen after 24 hours. (c) Efficient infection of resting CD4 T cells by three independent HIV LV particles indicated by mCherry transgene expression. Cells expressing mCherry remained viable (Supplementary Fig. 2) and negative for CD25/CD69 activation markers as indicated. (d) Fold increase of HIV LV infection after spinoculation with Vpx-VLPs as observed in CD4 T cells from peripheral blood, tonsil and spleen. (e) A marked decrease of surface CD4 expression in blood CD4 T cells productively infected with shCD4 HIV LV particles. (f) Reduced intracellular NLRP3 mRNA and protein expression in mCherry-positive CD4 T cells after infection with shNLRP3 HIV LV particles. Note that CD4 and NLRP3 expression were not reduced in mCherry-negative CD4 T cell populations. These data are representative of four independent experiments performed using CD4 T cells from peripheral blood, tonsil and spleen from four different donors.
Figure 2
Figure 2
Modulating specific CD4 T cells responses by HIV LV (a, b) Infection of shCD4 HIV LV impedes HIV-1 fusion to resting CD4 T cells isolated from blood. To test the ability of HIV-1 to fuse with infected cells, we used an HIV virion-based fusion assay that measures β-lactamase (BlaM) activity delivered to target cells upon the fusion of X4-tropic NL4-3 strain of HIV-1 containing BlaM fused to the Vpr protein (BlaM-Vpr-NL4-3). Inhibition of fusion was specifically observed in the mCherry-positive (a), but not in the mCherry-negative cells (b) residing in the same cultures. (c) Infection of shNLRP3 HIV LV blocks caspase-1 activation in tonsillar CD4 T cells treated with nigericin. (d) Infections with shNLRP3 HIV LV prevent caspase-1-mediated death of mCherry-positive CD4 T cells treated with nigericin. Inhibition of cell death was not inhibited by mCherry-positive Scramble HIV LV.

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